The candidate is currently a Research Fellow in the laboratory of Dr. Keji Zhao at NHLBI. The proposal describes a research and training program that will support the goal of transitioning the proposed research to an independent laboratory. The Career Development Award will provide a platform for continued training at NIH/NHLBI in support of the projects described in this proposal. Specific activities that will be supported include the formation of an Advisory Committee consisting of several scientists who have extensive scientific experience in fields related to work proposed in this application. These advisors also have significant experience in mentoring postdocs, of which many have obtained independent investigator positions in academia. This panel will be helpful in guiding my training and career goals. The mentored phase of the Career Development Plan will consist of continued training in bioinformatics and computational analysis, which is essential for application of next-generation sequencing technologies (e.g. RNA- Seq, ChIP-Seq), experimental training in genome integrity and cancer biology at the NIH to support the aims presented in this application, and development of mentorship skills through training of students. Specific mentoring activities during the Intramural phase will include training and mentoring a postbaccalaureate student in the scientific method including teaching hypothesis formation, experimental design, data analysis, and interpretation, as well as communication skills through public presentation of results. I will also work directly with a bioinformatics postdoc in Dr. Zhaos lab to develop skills in addressing biological questions through the use of computational biology. The intramural phase of this proposal will allow continued development of skills required to become a successful investigator such as communication skills, including presentation of results obtained from experiments proposed in the Research Plan at local and national conferences and meetings on topics related to the work described in this proposal. The goal of the research proposal is to investigate the role of SMYD5, a histone H4 lysine 20 histone methyltransferase, in regulating heterochromatin formation and genome stability in ES cells and during development. Dysregulation of heterochromatin leads to several human diseases including cancer and neurological disorders such as Friedreich's ataxia, Angelman syndrome, and Prader-Willi syndrome, and facioscapulohumeral muscular dystrophy. Heterochromatin plays a critical role in gene expression during development and differentiation, and is also involved in maintaining genome integrity by stabilizing repetitive DNA sequences throughout the genome. H4K20 methylation marks have been implicated in having diverse cellular functions including the formation of heterochromatin, gene regulation and repression of transcription, chromosome condensation, and genome stability. I recently discovered that SMYD5 is a novel mammalian H4K20 methyltransferase that deposits H4K20me3 marks at LTR/LINE repetitive DNA elements. Loss of SMYD5 results in decreased levels of heterochromatin constituents and causes chromosome aberrations and cellular transformation during in vitro ES cell differentiation. The expression signature of SMYD5-depleted transformed cells is correlated with a number of human cancers and can predict patient survival outcome. Despite these preliminary findings, our understanding about the role of SMYD5 in regulating heterochromatin formation and maintenance, and genome stability, is very limited. The experiments described in this proposal will clarify the role of SMYD5 in promoting chromatin compaction and controlling genome stability in ES cells and during development. I hypothesize that SMYD5 promotes chromatin compaction by regulating H4K20me3 levels and by recruiting heterochromatin proteins. I propose to define the role for SMYD5 in regulating heterochromatin formation by evaluating the chromatin accessibility of SMYD5 depleted ES cells and by investigating interactions between SMYD5 and heterochromatin proteins and long non-coding RNAs (lncRNAs) (Aim 1). I also hypothesize that SMYD5 acts as a tumor suppressor by regulating genome stability. I propose to cross SMYD5 knockout mice (generated for this project) with Suv420h2 deficient mice (another H4K20me3 methyltransferase) to further reduce H4K20me3 levels, and to evaluate tumor formation and chromosomal aberrations in cells from mutant mice (Aim 2). I also hypothesize that SMYD5 regulates gene expression in ES cells by silencing LTR elements nearby known genes, by interacting with transcriptional regulators, and by regulating a novel bivalent chromatin structure marked by co-occupancy of activating (H3K4me3) and repressing (H4K20me3) histone marks (Aim 3). Altogether, these studies will provide greater insight into the epigenetic role of a novel histone methyltransferase, SMYD5, in regulating heterochromatin formation and genome stability in ES cells and during differentiation.